Emulsion compositions

ABSTRACT

An emulsion is useful in allowing a wide variety of gene products to be expressed via eukaryotic in vitro expression. The emulsion comprises a silicone based surfactant, a hydrophobic phase and a hydrophilic phase; wherein the hydrophilic phase comprises a plurality of compartments containing a functional in vitro eukaryotic expression system.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.10/844,720, filed May 13, 2004, now U.S. Pat. No. 7,429,467 which is acontinuation of International Application No. PCT/GB02/05216, filed Nov.18, 2002, which claims the priority of Great Britain application GB0127564.3, filed Nov. 16, 2001, the entireties of which are herebyincorporated by reference.

FIELD OF THE INVENTION

The present invention relates, inter alia, to emulsions suitable forcompartmentalisation of transcription/translation reactions and methodsusing such emulsions. In particular, the emulsions are useful forcompartmentalisation of in vitro eukaryotic transcription/translationreactions.

BACKGROUND TO THE INVENTION

Compartnentalisation methods based on water-in-oil emulsions haverecently been developed for use in repertoire selection methods (Tawfik& Griffiths 1998, Ghadessy et al 2001). Compartmentalisation segregatesindividual genes and their encoded products (delivered either via cells(Ghadessy et al, 2001) or expressed in situ (Tawfik & Griffiths, 1998))into discrete, physically separate aqueous compartments, thus ensuringthe linkage of genotype and phenotype during the selection process.

WO99/02671. (which is incorporated herein by reference) describes amethod for isolating one or more genetic elements encoding a geneproduct having a desired activity. Genetic elements are firstcompartmentalised into microcapsules, which are preferably formed byemulsification, and are then transcribed and/or translated to producetheir respective gene products (RNA or protein) within themicrocapsules. Alternatively, the genetic elements may be containedwithin microcapsules of the emulsion and transcription and/ortranslation (expression) of the gene product can take place within usingthe cellular machinery. Genetic elements that produce a gene producthaving a desired activity can be subsequently sorted. For example, insome cases sorting may be due to the desired activity inducing a changein the microcapsule. In other cases sorting may be due to the desiredactivity inducing a change in the genetic element.

The method disclosed in WO99/02671 works well with bacteria. Althoughthe cellular subcompartmentalisation approach could in principle beextended to include eukaryotic cells, e.g. yeast, insect or mammaliancells, for some applications it would be desirable to provide in situexpression directly in microcapsules using an in vitro eukaryotictranscription/translation system.

Previous successful expression of a prokaryotic enzyme, Hae methylase,has been reported using bacterial S30 extracts in emulsion (Tawfik D. &Griffiths A. D. 1998). However, such methods are not suitable for someproteins of interest, for example large multi-domain proteins andribonucleoproteins which frequently cannot be expressed in functionalform using bacterial extracts.

Thus it can be seen that a method which enables in situ expressiondirectly in microcapsules using an in vitro eukaryotictranscription/translation system would provide a contribution to theart.

SUMMARY OF THE INVENTION

Accordingly, in a first aspect of the present invention, there isprovided an emulsion comprising a surfactant, a hydrophobic phase and ahydrophilic phase comprising a plurality of microcapsules containing afunctional in vitro eukaryotic expression system, wherein the surfactantis a chemically inert silicone-based surfactant.

In developing such a method, the present inventors have encounteredrepeated problems in maintaining efficient transcriptional/translationalability of an eukaryotic system, in particular the rabbit reticulocytelysate system, when in emulsion. As described below, a number ofmodifications to emulsion compositions used with prokaryotic expressionsystems were unsuccessful in conferring such ability. However, it wassurprisingly found that, when chemically inert silicone-basedsurfactants were employed in the emulsion composition, the efficiency oftranscription of the eukaryotic transcription/translation system wasmarkedly improved.

The emulsion allows the linkage of genotype and phenotype due tocompartmentalisation of the eukaryotic expression system, whilstavoiding many of the disadvantages of cell-based systems. Thus, forexample, it allows gene products to be obtained without the need forextraction or secretion from cells.

Accordingly, in a second aspect of the invention, there is provided amethod of isolating one or more genetic elements encoding a gene producthaving a desired activity, comprising the steps of:

-   -   (a) compartinentalising the genetic elements into microcapsules        formed from an emulsion of the invention    -   (b) expressing the genetic elements to produce their respective        gene products within the microcapsules;    -   (c) sorting the genetic elements which produce gene product(s)        having the desired activity.

In a third aspect, the invention provides a method for preparing a geneproduct, comprising the steps of

-   -   (a) preparing a genetic element encoding the gene product;    -   (b) compartmentalising genetic elements into microcapsules        formed from an emulsion of the invention;    -   (c) expressing the genetic elements to produce their respective        gene products within the microcapsules;    -   (d) sorting the genetic elements which produce the gene        product(s) having the desired activity; and    -   (e) expressing the gene product having the desired activity.

In accordance with this aspect of the invention, step (a) preferablycomprises preparing a repertoire of genetic elements, wherein eachgenetic element encodes a potentially differing gene product.Repertoires may be generated by conventional techniques, such as thoseemployed for the generation of libraries intended for selection bymethods such as phage display. Gene products having the desired activitymay be selected from the repertoire, according to the present invention.

A fourth aspect of the invention provides a product selected using themethod of the second aspect of the invention or prepared according tothe third aspect of the invention. As used in this context, a “product”may refer to a gene product selected or prepared according to theseaspects, or the genetic element (or genetic information comprisedtherein).

In a fifth aspect, the invention provides a method for screening acompound or compounds capable of modulating the activity of a geneproduct, comprising the steps of:

-   -   (a) preparing a repertoire of genetic elements encoding gene        product;    -   (b) compartmentalising the genetic elements into microcapsules        formed from an emulsion of the invention;    -   (c) expressing the genetic elements to produce their respective        gene products within the microcapsules;    -   (d) sorting the genetic elements which produce the gene        product(s) having the desired activity; and    -   (e) contacting a gene product having the desired activity with        the compound or compounds and monitoring the modulation of an        activity of the gene product by the compound or compounds.

In the context of the present invention, a surfactant is considered tobe “chemically inert” if it is substantially free of oxidating species,such as peroxides and aldehydes, and protein denaturing species.Surfactants having 40%, preferably 50%, more preferably 60%, 70%, 80%,90%, 95%, or 98% less oxidating species and protein denaturing speciesthan either one of conventional sorbitan monooleate (Span™80; ICI) andpolyoxyethylenesorbitan monooleate (Tween™ 80; ICI) emulsifiers areconsidered to be “substantially free” of oxidating species anddenaturing species.

The terms “isolating”, “sorting” and “selecting”, as well as variationsthereof, are used herein. Isolation, according to the present invention,refers to the process of separating an entity from a heterogeneouspopulation, for example a mixture, such that it is free of at least onesubstance with which it was associated before the isolation process. Ina preferred embodiment, isolation refers to purification of an entityessentially to homogeneity. Sorting of an entity refers to the processof preferentially isolating desired entities over undesired entities. Inas far as this relates to isolation of the desired entities, the terms“isolating” and “sorting” are equivalent. The method of the presentinvention permits the sorting of desired genetic elements from pools(libraries or repertoires) of genetic elements which contain the desiredgenetic element. Selecting is used to refer to the process (includingthe sorting process) of isolating an entity according to a particularproperty thereof.

In preferred embodiments of the methods of the invention, the sorting ofgenetic elements may be performed in one of essentially four techniques,details of which are given in WO99/02671.

-   (I) In a first embodiment, the microcapsules are sorted according to    an activity of the gene product or a derivative thereof which makes    the microcapsule detectable as a whole. Accordingly, the invention    provides a method according to the second aspect of the invention    wherein a gene product with the desired activity induces a change in    the microcapsule, or a modification of one or more molecules within    the microcapsule, which enables the microcapsule containing the gene    product and the genetic element encoding it to be sorted. In this    embodiment, therefore, the microcapsules are physically sorted from    each other according to the activity of the gene product(s)    expressed from the genetic element(s) contained therein, which makes    it possible selectively to enrich for microcapsules containing gene    products of the desired activity.-   (II) In a second embodiment, the genetic elements are sorted    following pooling of the microcapsules into one or more common    compartments. In this embodiment, a gene product having the desired    activity modifies the genetic element which encoded it (and which    resides in the same microcapsule) in such a way as to make it    selectable in a subsequent step. The reactions are stopped and the    microcapsules are then broken so that all the contents of the    individual microcapsules are pooled. Selection for the modified    genetic elements enables enrichment of the genetic elements encoding    the gene product(s) having the desired activity. Accordingly, the    invention provides a method according to the second aspect of the    invention, wherein in step (b) the gene product having the desired    activity modifies the genetic element encoding it to enable the    isolation of the genetic element. It is to be understood, of course,    that modification may be direct, in that it is caused by the direct    action of the gene product on the genetic element, or indirect, in    which a series of reactions, one or more of which involve the gene    product having the desired activity, leads to modification of the    genetic element.-   (III) In a third embodiment, the genetic elements are sorted    following pooling of the microcapsules into one or more common    compartments. In this embodiment, a gene with a desired activity    induces a change in the microcapsule containing the gene product and    the genetic element encoding it. This change, when detected,    triggers the modification of the gene within the microcapsule. The    reactions are stopped and the microcapsules are then broken so that    all the contents of the individual microcapsules are pooled.    Selection for the modified genetic elements enables enrichment of    the genetic elements encoding the gene product(s) having the desired    activity. Accordingly the invention provides a method according to    the second aspect of the invention, where in step (b) the gene    product having the desired activity induces a change in the    microcapsule which is detected and triggers the modification of the    genetic element within the microcapsule so as to allow its    isolation. It is to be understood that the detected change in the    microcapsule may be caused by the direct action of the gene product,    or indirect action, in which a series of reactions, one or more of    which involve the gene product having the desired activity leads to    the detected change.-   (IV) In a fourth embodiment, the genetic elements may be sorted by a    multi-step procedure, which involves at least two steps, for    example, in order to allow the exposure of the genetic elements to    conditions which permit at least two separate reactions to occur. As    will be apparent to a persons skilled in the art, the first    microencapsulation step of the invention must result in conditions    which permit the expression of the genetic elements—be it    transcription, transcription and/or translation, replication or the    like. Under these conditions, it may not be possible to select for a    particular gene product activity, for example because the gene    product may not be active under these conditions, or because the    expression system contains an interfering activity. The invention    therefore provides a method according to the second aspect of the    present invention, wherein step (b) comprises expressing the genetic    elements to produce their respective gene products within the    microcapsules, linking the gene products to the genetic elements    encoding them and isolating the complexes thereby formed. This    allows for the genetic elements and their associated gene products    to be isolated from the microcapsules before sorting according to    gene product activity takes place. In a preferred embodiment, the    complexes are subjected to a further compartmentalisation step prior    to isolating the genetic elements encoding a gene product having the    desired activity. This further compartmentalisation step, which    advantageously takes place in microcapsules, permits the performance    of further reactions, under different conditions, in an environment    where the genetic elements and their respective gene products are    physically linked. Eventual sorting of genetic elements may be    performed according to embodiment (I), (II) or (III) above.

The “secondary encapsulation” may also be performed with geneticelements linked to gene products by other means, such as by phagedisplay, polysome display, RNA-peptide fusion or lac repressor peptidefusion.

The selected genetic element(s) may also be subjected to subsequent,possibly more stringent rounds of sorting in iteratively repeated steps,reapplying the method of the invention either in its entirety or inselected steps only. By tailoring the conditions appropriately, geneticelements encoding gene products having a better optimised activity maybe isolated after each round of selection.

Additionally, the genetic elements isolated after a first round ofsorting may be subjected to mutagenesis before repeating the sorting byiterative repetition of the steps of the method of the invention as setout above. After each round of mutagenesis, some genetic elements willhave been modified in such a way that the activity of the gene productsis enhanced.

Moreover, the selected genetic elements can be cloned into an expressionvector to allow further characterisation of the genetic elements andtheir products. A multitude of suitable vectors are known to the personskilled in the art. The vectors may be, for example, virus, plasmid orphage vectors provided with an origin of replication, optionally apromoter for the expression of the genetic element and optionally aregulator of the promoter. The vectors may contain a selectable markergene, for example the neomycin resistance gene for a mammalian vector.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows emulsions formed by mixing oil-phase with rabbitreticulocyte lysate (RRL) for 1.5 min and photographed in visualspectrum after 15 min incubation at 30° C. From left to right: 1) CSRmix, 2) CSR mix+DTT, 3) 1.5% Span80 in mineral oil, 4) 1.5% Span80 inmineral oil+DTT, 5) 4% Abil EM90, 6) 4% Abil EM90+DTT

FIG. 2 shows a 4% Abil EM90 in mineral oil emulsion imaged inphase-contrast mode. The distance between the bars is 10 M

FIG. 3 shows the results of telomerase expression:

Panel A (Non-emulsified): Lane 1, hTERT (75 ng); Lane 2, hTR (75 ng);Lane 3, hTERT+hTR; Lane 4, No construct; Lane 5, Tandem WT expressionconstruct (150 ng) Lane 6, Tandem deletion mutant construct (150 ng)

Panel B: In emulsion, lane annotation same as in panel A.

DETAILED DESCRIPTION

Emulsions

Emulsions are heterogeneous systems of two immiscible liquid phases withone of the phases dispersed in the other as droplets of microscopic orcolloidal size (Becher, 1957; Sherman, 1968; Lissant, 1984). Emulsionsof the invention must enable the formation of microcapsules.

Emulsions may be produced from any suitable combination of immiscibleliquids. The emulsion of the present invention has a hydrophilic phase(containing the biochemical components) as the phase present in the formof finely divided droplets (the disperse, internal or discontinuousphase) and a hydrophobic, immiscible liquid (an ‘oil’) as the matrix inwhich these droplets are suspended (the nondisperse, continuous orexternal phase). Such emulsions are termed ‘water-in-oil’ (W/O). Thishas the advantage that the entire aqueous phase containing thebiochemical components is compartnentalised in discreet droplets (theinternal phase). The external phase, being a hydrophobic oil, generallycontains none of the biochemical components and hence is inert.

Creation of an emulsion generally requires the application of mechanicalenergy to force the phases together. There are a variety of ways ofdoing this which utilise a variety of mechanical devices, includingstirrers (such as magnetic stir-bars, propeller and turbine stirrers,paddle devices and whisks), homogenisers (including rotor-statorhomogenisers, high-pressure valve homogenisers and jet homogenisers),colloid mills, ultrasound and ‘membrane emulsification’ devices (Becher,1957; Dickinson, 1994).

Desirably, the emulsion is stable during incubation at 30° C. for atleast one hour. In some cases it is preferred that it also be stable athigher temperatures, especially if thermal cycling is used during PCR orother amplification procedures.

Surfactants

Emulsions of the invention are stabilised by addition of one or moresurface-active agents (surfactants). These surfactants are termedemulsifying agents and act at the water/oil interface to prevent (or atleast delay) separation of the phases. Many oils and many emulsifierscan be used for the generation of water-in-oil emulsions; a recentcompilation listed over 16,000 surfactants, many of which are used asemulsifying agents (Ash and Ash, 1993).

However, as described in the examples, surfactants conventionally usedin emulsions applications such as non-ionic surfactants (Schick, 1966),for example, sorbitan monooleate (Span™80; ICI) andpolyoxyethylenesorbitan monooleate (Tween™ 80; ICI) are not suitable forefficient in vitro eukaryotic expression with the rabbit reticulocytelysate

However, as described herein, such expression may be maintained whenchemically inert silicone-based surfactants are used.

Preferably, the chemically inert silicone-based surfactant of theemulsion is a silicone copolymer.

More preferably the surfactant comprises apolysiloxane-polycetyl-polyethylene glycol copolymer (Cetyl DimethiconeCopolyol) e.g. Abil.® EM90 (Goldschmidt).

The chemically inert silicone-based surfactant may be provided as thesole surfactant in the emulsion composition or may be provided as one ofseveral surfactants. For example a mixture of different surfactants maybe used.

In preferred embodiments, the surfactant is provided at a v/vconcentration in the oil phase of the emulsion of 0.5 to 20%, preferably1 to 10%, more preferably 3-5%.

In a highly preferred embodiment, the surfactant is provided at a v/vconcentration in the oil phase of 4%.

In a highly preferred embodiment of the invention the emulsion is madeby adding an aqueous phase dropwise to an oil phase in the presence of asurfactant comprising about 3-5% (v/v)polysiloxane-polycetyl-polyethylene glycol copolymer in mineral oil,preferably at a ratio of oil:water phase of 2.5:1.

The surfactant may be present initially with the hydrophobiccomposition. A composition comprising the surfactant and the hydrophobiccomposition therefore represents a further aspect of the presentinvention.

Alternatively, the surfactant may be added at a later stage, e.g. duringor following the mixing of the hydrophobic and hydrophilic phases.

In this embodiment the surfactant may be provided in a kit, togetherwith a hydrophobic composition. The kit may optionally also include aeukaryotic expression system such as rabbit reticulocyte lysate and/or adevice for mixing the hydrophobic and hydrophilic phases. It may furtherinclude instructions for use in providing an emulsion of the presentinvention

Alternatively, the surfactant may be present initially with thehydrophilic composition.

Microcapsules

The term “microcapsule” is synonymous with “compartment” and the termsare used interchangeably. In essence, a microcapsule is an artificialcompartment whose delimiting borders restrict the exchange of thecomponents of the molecular mechanisms described herein which allow thesorting of genetic elements according to the function of the geneproducts which they encode.

The microcapsules formed by the emulsion require appropriate physicalproperties to allow the working of the invention. In particular, thecontents of each microcapsule must be isolated from the contents ofsurrounding microcapsules.

Preferably, the microcapsules used in the methods of the presentinvention will be capable of being produced in very large numbers, andthereby to compartmentalise a library of genetic elements which encodesa repertoire of gene products. The function of the microcapsule is toenable co-localisation of the nucleic acid and the correspondingpolypeptide encoded by the nucleic acid. This is preferably achieved bythe ability of the microcapsule to substantially restrict diffusion oftemplate and product strands to other microcapsules.

Second, the methods of the present invention require that there are onlya limited number of genetic elements per microcapsule formed in theemulsion. This ensures that the gene product of an individual geneticelement will be isolated from other genetic elements. Thus, couplingbetween genetic element and gene product will be highly specific. Theenrichment factor is greatest with on average one or fewer geneticelements per microcapsule, or two or more copies of a single geneticelement per microcapsule, the linkage between nucleic acid and theactivity of the encoded gene product being as tight as is possible,since the gene product of an individual genetic element will be isolatedfrom the products of all other genetic elements. However, even if thetheoretically optimal situation of, on average, a single genetic elementor less per microcapsule is not used, a ratio of 5, 10, 50, 100 or 1000or more genetic elements per microcapsule may prove beneficial insorting a large library. Subsequent rounds of sorting, including renewedencapsulation with differing genetic element distribution, will permitmore stringent sorting of the genetic elements. Preferably, there is asingle genetic element, or fewer, per microcapsule.

Thirdly, the formation and the composition of the microcapsules must notabolish the function of the machinery for the expression of the geneticelements and the activity of the gene products using eukaryotic in vitroexpression systems.

Consequently, the microcapsules formed by the emulsion must fulfil thesethree requirements. The appropriate emulsion may vary depending on theprecise nature of the requirements in each application of the invention,as will be apparent to the skilled person.

The preferred microcapsule size will vary depending upon the preciserequirements of any individual selection process that is to be performedaccording to the present invention. In all cases, the principleconsideration is the need for required concentration of components inthe individual compartments to achieve efficienttranscription/translation. This may be balanced with other requirementswhich may relate to gene library size, enrichment, or sortingprocedures.

If small microcapsules are provided, a very large number of discretemicrocapsules can be provided within a small volume of emulsion.Furthermore the provision of small microcapsules increases selectivityby increasing the likelihood that only one type of gene product will beproduced within a given microcapsule. It is therefore preferred that onaverage no more than one genetic elements that is to be transcribedand/or translated is present per compartment in order to keep thelinkage between nucleic acid and the activity of encoded gene product astight as is possible. However, even if the theoretically optimalsituation of, on average, a single genetic element or less permicrocapsule is not used, a ratio of 5, 10, 50, 100 or 1000 or moregenetic elements per microcapsule may prove beneficial in sorting alarge library. Subsequent rounds of sorting, including renewedencapsulation with differing genetic element distribution, will permitmore stringent sorting of the genetic elements. Preferably, there is onaverage no more than a single genetic element per microcapsule.

Desirably, the mean volume of the microcapsules is less that 5.2×10⁻¹⁶m³, (corresponding to a spherical microcapsule of diameter less than 10μm), more desirably less than 6.5×10⁻¹⁷ m³ (corresponding to a sphericalmicrocapsule of diameter less than 5 μm), still more desirably about4.2×10⁻¹⁸ m³ (corresponding to a spherical microcapsule of diameter ofapproximately 2 μm).

The effective DNA or RNA concentration in microcapsules may beartificially increased by various methods that will be well-known tothose versed in the art. These include, for example, the addition ofvolume excluding chemicals such as polyethylene glycols (PEG) and avariety of gene amplification techniques, including transcription usingRNA polymerases including those from bacteria such as E. coli (Roberts,1969; Blattner and Dahlberg, 1972; Roberts et al., 1975; Rosenberg etal., 1975, eukaryotes e.g. (Weil et al., 1979; Manley et al., 1983) andbacteriophage such as T7, T3 and SP6 (Melton et al., 1984); thepolymerase chain reaction (PCR) (Saiki et al., 1988); Qβ replicaseamplification (Miele et al., 1983; Cahill et al., 1991; Chetverin andSpirin, 1995; Katanaev et al., 1995); the ligase chain reaction (LCR)(Landegren et al., 1988; Barany, 1991); and self-sustained sequencereplication system (Fahy et al., 1991) and strand displacementamplification (Walker et al., 1992). Gene amplification techniquesadvantageously using thermal cycling (such as PCR and LCR) may be usedif the emulsions and the in vitro transcription or coupledtranscription-translation systems are thermostable.

Increasing the effective local nucleic acid concentration enablesmicrocapsules to be used more effectively. Thus microcapsules havingvolumes of up to only about 5.2×10⁻¹⁶ m³ (corresponding to a sphere ofdiameter 10 μm) can be used for many purposes, although of coursemicrocapsules with larger volumes can be used, if desired.

The microcapsule size must be sufficiently large to accommodate all ofthe required components of the biochemical reactions that are needed tooccur within the microcapsule. For example, in vitro, both transcriptionreactions and coupled transcription-translation reactions often requirea total nucleoside triphosphate concentration of about 2 mM.

It can also be noted that, in order to transcribe a gene to a singleshort RNA molecule of 500 bases in length, this would require a minimumof 500 molecules of nucleoside triphosphate per microcapsule (8.33×10⁻²²moles). In order to constitute a 2 mM solution, this number of moleculesmust be contained within a microcapsule of volume 4.17×10⁻¹⁹ litres(4.17×10⁻²² m³), which, if spherical, would have a diameter of 93 nm.

Furthermore, particularly in the case of reactions involvingtranslation, it is to be noted that the eukaryotic ribosomes necessaryfor the translation to occur are themselves approximately 30 nm indiameter. Hence, the preferred lower limit for microcapsules is adiameter of approximately 0.1 μm (100 nm).

Therefore, the microcapsule volume is preferably of the order of between5.2×10⁻²² m³ and 5.2×10⁻¹⁶ m³ corresponding to a sphere of diameterbetween 0.1 μm and 10 μm, more preferably of between about 5.2×10⁻¹⁹ m³and 6.5×10⁻¹⁷ m³ (1 μm and 5 μm).

Although small microcapsules are preferred for certain applications,such as the methods disclosed in WO99/02671, it is important to notethat the present invention is in no way limited to the provision ofsmall microcapsules and that transcription and translation systems alsofunction in large microcapsules.

The size of emulsion microcapsules may be varied simply by tailoring theemulsification conditions used to form the emulsion according torequirements of the selection system.

Compartment size may be varied (within limits of emulsion stability andinactivation of RRL) by 1) increased mixing time, 2) different W/Oratios, 3) different concentrations of surfactant.

The size distribution of microcapsules in emulsions can be determined byany method known to skilled person. For example, the size distributionmay be assessed using laser diffraction (e.g. using a Coulter LS230Particle Size Analyser) or by microscopic examination.

Expression

“Expression”, as used herein, is used in its broadest meaning, tosignify that a nucleic acid contained in the genetic element isconverted into its gene product. Thus, where the nucleic acid is DNA,expression refers to the transcription of the DNA into RNA; where thisRNA codes for protein, “expression” may also refer to the translation ofthe RNA into protein. Where the nucleic acid is RNA, “expression” mayrefer to the replication of this RNA into further RNA copies, thereverse transcription of the RNA into DNA and optionally thetranscription of this DNA into further RNA molecule(s), as well asoptionally the translation of any of the RNA species produced intoprotein. Preferably, therefore, “expression” is performed by one or moreprocesses selected from the group consisting of transcription, reversetranscription, replication and translation.

“Expression” of the genetic element may thus be directed into eitherDNA, RNA or protein, or a nucleic acid or protein containing unnaturalbases or amino acids (the gene product) within the microcapsule of theinvention, so that the gene product is confined within the samemicrocapsule as the genetic element.

Expression Systems

Any appropriate in vitro eukaryotic expression system can be used in theemulsion of the invention provided that it includes components neededfor transcription and/or translation. If glycosylation is desired thenone or more glycosylases can also be present, as appropriate, in orderto achieve a desired glycosylation pattern.

The expression system may, for example, comprise all or part of a celllysate or extract.[For example wheat germ extract may be used (Andersonet al., 1983) and is available commercially from Promega. Desirably,however, the cell lysate is a mammalian cell lysate. It may be areticulocyte lysate. A preferred reticulocyte lysate is a rabbitreticulocyte lysate (an “RRL”). The RRL system is well characterised(see e.g. Pelham and Jackson, 1976) and is available commercially fromPromega.

However, any convenient eukaryotic expression system prepared from othercell extracts may be used. The extracts should contain all thecomponents required for translation of RNA (e.g. ribosomes, tRNAs,aminoacyl-tRNA synthetases, initiation, elongation and terminationfactors etc with the extracts preferably supplemented with amino acids,ATP, GTP, creatine phosphate and creatine phosphokinase, otherco-factors such as Mg²⁺.

By using emulsions of the present invention it is possible to obtainappreciable expression of a genetic element and/or activity of a geneproduct using an in vitro eukaryotic expression system present within anemulsion. Expression and/or activity is desirably at a level of at least1% of that of the gene product achievable with the expression systemprior to formation of the emulsion. More preferably it is at least 10%,20% or 30% of said level and/or activity.

Genetic Elements

A “genetic element” is a molecule or molecular construct comprising anucleic acid. The genetic elements of the present invention may compriseany nucleic acid (for example, DNA, RNA or any analogue, natural orartificial, thereof).

Nucleic acids encoding any appropriate gene product can be used in thein vitro transcription and/or translation systems. In addition to codingsequences, the nucleic acids may, for example, comprise promoters,operators, enhancers, translational and transcriptional initiation andtermination sequences, polyadenylation sequences, splice sites, upstreamand downstream regulatory regions, etc., as required for transcriptionand/or translation. In some cases it may be preferred to use inducibleand/or temperature sensitive promoters in order to ensure expressionoccurs only at a particular stage.

The nucleic acid component of the genetic element may moreover belinked, covalently or non-covalently, to one or more molecules orstructures, including polypeptides, peptides, chemical entities andgroups, solid-phase supports such as magnetic beads, and the like. Inthe methods of the invention, these structures or molecules can bedesigned to assist in the sorting and/or isolation of the geneticelement encoding a gene product with the desired activity.

As will be apparent from the following, in many cases the polypeptide orother molecular group or construct is a ligand or a substrate whichdirectly or indirectly binds to or reacts with the gene product in orderto tag the genetic element. This allows the sorting of the geneticelement on the basis of the activity of the gene product.

The ligand or substrate can be connected to the nucleic acid by avariety of means that will be apparent to those skilled in the art (see,for example, Hermanson, 1996). Any tag will suffice that allows for thesubsequent selection of the genetic element. Sorting can be by anymethod which allows the preferential separation, amplification orsurvival of the tagged genetic element. Examples include selection bybinding (including techniques based on magnetic separation, for exampleusing Dynabeads™), and by resistance to degradation (for example bynucleases, including restriction endonucleases).

One way in which the nucleic acid molecule may be linked to a ligand orsubstrate is through biotinylation. This can be done by PCRamplification with a 5′-biotinylation primer such that the biotin andnucleic acid are covalently linked. A biotinylated nucleic acid may becoupled to a polystyrene microbead (0.035 to 0.2 μm in diameter) that iscoated with avidin or streptavidin, that will therefore bind the nucleicacid with very high affinity. This bead can be derivatised withsubstrate or ligand by any suitable method such as by addingbiotinylated substrate or by covalent coupling.

Alternatively, a biotinylated nucleic acid may be coupled to avidin orstreptavidin complexed to a large protein molecule such as thyroglobulin(669 Kd) or ferritin (440 Kd). This complex can be derivatised withsubstrate or ligand, for example by covalent coupling to the -aminogroup of lysines or through a non-covalent interaction such asbiotin-avidin. The substrate may be present in a form unlinked to thegenetic element but containing an inactive “tag” that requires a furtherstep to activate it such as photoactivation (e.g. of a “caged” biotinanalogue, (Sundberg et al., 1995; Pirrung and Huang, 1996)). Thecatalyst to be selected then converts the substrate to product. The“tag” could then be activated and the “tagged” substrate and/or productbound by a tag-binding molecule (e.g. avidin or streptavidin) complexedwith the nucleic acid. The ratio of substrate to product attached to thenucleic acid via the “tag” will therefore reflect the ratio of thesubstrate and product in solution.

An alternative is to couple the nucleic acid to a product-specificantibody (or other product-specific molecule). In this scenario, thesubstrate (or one of the substrates) is present in each microcapsuleunlinked to the genetic element, but has a molecular “tag” (for examplebiotin, DIG or DNP). When a catalyst to be selected converts thesubstrate to product, the product retains the “tag” and is then capturedin the microcapsule by the product-specific antibody. In this way thegenetic element only becomes associated with the “tag” when it encodesor produces an enzyme capable of converting substrate to product.

When all reactions are stopped and the microcapsules are combined, thegenetic elements encoding active enzymes can be enriched using anantibody or other molecule which binds, or reacts specifically with the“tag”. Although both substrates and product have the molecular tag, onlythe genetic elements encoding active gene product will co-purify.

In a highly preferred application, the methods of the present inventionare useful for sorting libraries of genetic elements. The inventionaccordingly provides a method according to preceding aspects of theinvention, wherein the genetic elements are isolated from a library ofgenetic elements encoding a repertoire of gene products. Herein, theterms “library”, “repertoire” and “pool” are used according to theirordinary signification in the art, such that a library of geneticelements encodes a repertoire of gene products. In general, librariesare constructed from pools of genetic elements and have properties whichfacilitate sorting.

Initial selection of a genetic element from a genetic element libraryusing the present invention will in most cases require the screening ofa large number of variant genetic elements. Libraries of geneticelements can be created in a variety of different ways, including thefollowing.

Pools of naturally occurring genetic elements can be cloned from genomicDNA or cDNA (Sambrook et al., 1989); for example, phage antibodylibraries, made by PCR amplification repertoires of antibody genes fromimmunised or unimmunised donors have proved very effective sources offunctional antibody fragments (Winter et al., 1994; Hoogenboom, 1997).Libraries of genes can also be made by encoding all (see for exampleSmith, 1985; Parmley and Smith, 1988) or part of genes (see for exampleLowman et al., 1991) or pools of genes (see for example Nissim et al.,1994) by a randomised or doped synthetic oligonucleotide. Libraries canalso be made by introducing mutations into a genetic element or pool ofgenetic elements ‘randomly’ by a variety of techniques in vivo,including; using ‘mutator strains’, of bacteria such as E. coli mutD5(Liao et al., 1986; Yamagishi et al., 1990; Low et al., 1996); using theantibody hypermutation system of B-lymphocytes (Yelamos et al., 1995).Random mutations can also be introduced both in vivo and in vitro bychemical mutagens, and ionising or UV irradiation (see Friedberg et al.,1995), or incorporation of mutagenic base analogues (Freese, 1959;Zaccolo et al., 1996). ‘Random’ mutations can also be introduced intogenes in vitro during polymerisation for example by using error-pronepolymerases (Leung et al., 1989).

Further diversification can be introduced by using homologousrecombination either in vivo (see Kowalczykowski et al., 1994) or invitro (Stemmer, 1994a; Stemmer, 1994b).

Gene Product

The present invention can thus be used to produce specific, desired geneproducts or to produce a range of diverse gene products (which may bepartially or wholly unknown) for screening purposes.

The term “gene product” is used herein in its broadest sense to includenot only polypeptides but also RNA gene products. Thus it includes theproducts of transcription alone, as well as the products of bothtranscription and translation.

Preferred gene products are polypeptides that occur naturally ineukaryotic cells (especially mammalian or human cells) but not inprokaryotic cells, or mutant forms of such polypeptides having one ormore amino acid changes relative to the wild type eukaryoticpolypeptide. Mutant forms are useful in generating diversity, e.g. forscreening purposes. However, if amino acid changes are made it ispreferred that there is a limited number of such changes, e.g. that lessthan 50, less than 25, less than 10 or less than 5 amino acids arechanged relative to a wild type polypeptide. Mutant forms may, forexample, act as antagonists or agonists of one or more of the biologicalactivities of a wild-type polypeptide. They may therefore be useful instudying structure-function relationships, in screening, in drugdevelopment programs, etc. Mutations can be introduced into nucleicacids by any appropriate method. For example, a nucleic acid sequenceincorporating a desired sequence change can be provided by site-directedmutagenesis. This can then be used to allow the expression of an RNA ora polypeptide having a corresponding change in its sequence.Alternatively, a nucleic acid may be synthesised to include a givenmutation. Mutations can also be provided by using mutagenic agentsand/or irradiation.

For certain applications the expression system may be used to express agene product useful in the replication, repair, maintenance orreplication of a nucleic acid of a eukaryotic cell, preferably of amamnialian or human cell. The gene product may therefore have activityas a polymerase, a reverse transcriptase, a ligase, or a telomerase, forexample. It may have more than one such activity. If it is involved innucleic acid repair, it may have proofreading activity.

Gene products can be produced in the compartments formed by thehydrophilic phase of the emulsion and containing the expression system.

Selection Procedures

The methods can be configured to select for RNA, DNA or protein geneproduct molecules with catalytic, regulatory or binding activity.

(i) Affinity Selection

In the case of selection for a gene product with affinity for a specificligand the genetic element may be linked to the gene product in themicrocapsule via the ligand. Only gene products with affinity for theligand will therefore bind to the genetic element itself and thereforeonly genetic elements that produce active product will be retained inthe selection step. In this embodiment, the genetic element will thuscomprise a nucleic acid encoding the gene product linked to a ligand forthe gene product.

In this embodiment, all the gene products to be selected contain aputative binding domain, which is to be selected for, and a commonfeature—a tag. The genetic element in each microcapsule is physicallylinked to the ligand. If the gene product produced from the geneticelement has affinity for the ligand, it will bind to it and becomephysically linked to the same genetic element that encoded it, resultingin the genetic element being ‘tagged’. At the end of the reaction, allof the microcapsules are combined, and all genetic elements and geneproducts pooled together in one environment. Genetic elements encodinggene products exhibiting the desired binding can be selected by affinitypurification using a molecule that specifically binds to, or reactsspecifically with, the “tag”.

In an alternative embodiment, genetic elements may be sorted on thebasis that the gene product, which binds to the ligand, merely hides theligand from, for example, further binding partners. In this eventuality,the genetic element, rather than being retained during an affinitypurification step, may be selectively eluted whilst other geneticelements are bound.

In an alternative embodiment, the invention provides a method accordingto the second aspect of the invention, wherein in step (b) the geneproducts bind to genetic elements encoding them. The gene productstogether with the attached genetic elements are then sorted as a resultof binding of a ligand to gene products having the desired activity. Forexample, all gene products can contain an invariant region which bindscovalently or non-covalently to the genetic element, and a second regionwhich is diversified so as to generate the desired binding activity.

Sorting by affinity is dependent on the presence of two members of abinding pair in such conditions that binding may occur. Any binding pairmay be used for this purpose. As used herein, the term binding pairrefers to any pair of molecules capable of binding to one another.Examples of binding pairs that may be used in the present inventioninclude an antigen and an antibody or fragment thereof capable ofbinding the antigen, the biotin-avidin/streptavidin pair (Savage et al.,1994), a calcium-dependent binding polypeptide and ligand thereof (e.g.calmodulin and a calmodulin-binding peptide (Stofko et al., 1992;Montigiani et al., 1996)), pairs of polypeptides which assemble to forma leucine zipper (Tripet et al., 1996), histidines (typicallyhexahistidine peptides) and chelated Cu²⁺, Zn²⁺ and Ni²⁺, (e.g. Ni-NTA;Hochuli et al., 1987), RNA-binding and DNA-binding proteins (Klug, 1995)including those containing zinc-finger motifs (Klug and Schwabe, 1995)and DNA methyltransferases (Anderson, 1993), and their nucleic acidbinding sites.

However, the usefulness of certain affinity based selection methods maybe limited. Especially with low to medium affinity interactions, thehalf-life of the interactions may often be too short compared with thetime required to detect the interactions.

Thus in one embodiment of the present invention, selection methods arebased on increasing the affinity of intermolecular interactions byallowing multivalent interactions to occur. The resulting apparentincrease in affinity gained by multivalent interaction, also termedavidity, is due to the fact that when two or more binding interactionstake place within the same molecular complex, there is only a very smalladditional entropic price to be paid for the second or furtherinteractions. This is because most degrees of freedom have already beenlost when binding through one binding site immobilised the multivalentligand. However, avidity can have particularly drastic effects on thedissociation kinetics (koff), as all interactions must be broken beforedissociation can take place.

Thus, in this embodiment, molecules that interact may be selected byallowing multivalent interactions to occur between the molecules, thusincreasing the stability of any complex formed as compared with acorresponding monovalent interaction.

Preferably, the first and/or second interacting molecules, e.g. the geneproduct and the ligand molecule to which it binds, have been modified toincrease their valency. Preferably, one or more reactive groups ispresent on the gene product or ligand molecules, or both, such that thegene product (and/or ligand) molecules associate with each other to formmultivalent gene product complexes and/or multivalent ligand moleculecomplexes. The multivalent complexes may then be selected for bindingaccording to the invention. Preferably, the reactive groups form acovalent bond on interaction.

The term “multivalent complex” as used herein means a molecular complexcomprising (i) at least two molecules of the gene product or at leastone molecule of a multivalent gene product and (ii) a multivalent ligandmolecule, wherein the at least two molecules of the gen product areinteracting with the ligand molecule or the at least one molecule of amultivalent gen product is interacting with the ligand molecule via atleast two valencies.

In such avidity selection procedures, the ligand molecule is preferablymultivalent. For example, where the ligand molecule is a polypeptide,the ligand molecule may be multivalent by virtue of being expressed as afusion protein to a third polypeptide which multimerises.

The gene product and/or ligand molecules may comprise a tag, for examplea biotin or a myc epitope tag or moiety, to assist in purifyingcomplexes formed between them, and/or to assist in recovering geneproduct and/or ligand molecules for analysis and identification.

The ligand molecule is typically a multimer, such as a dimer, or capableof forming a homomultimer under the reaction conditions used in themethods of the invention. Alternatively, the ligand molecule may be aheteromultimer. It is preferred to use ligand molecules which arepolypeptides that have been engineered to be multimeric but whoseconstituent subunits do not normally form multimers. This may beachieved by chemical linkage of two or more molecules to form acovalently linked multimer. Alternatively, in the case of ligandmoleculoes which are polypeptides and which bind to gene products whichare polypeptides, the molecules may be expressed as a fusion to a thirdpolypeptide, the third polypeptide forming homomultimers whicheffectively results in multimerisation of the fused ligand polypeptidesequence. An example of a suitable third polypeptide (referred to hereinas a “hook” polypeptide) is glutathione-S-transferase (GST), which formsdirners. Preferred hook polypeptides provide for good levels ofexpression of soluble products. In one embodiment, hook polypeptidesthat are suitable for secretion into the bacterial periplasm (e.g. GSTafter the removal of 3 surface Cys residues (Tudyka and Skerra, 1997)are preferred. The ligand molecule may be provided as a plurality ofligand molecules.

In a particularly preferred embodiments of the invention, the geneproducts are polypeptides encoded by first polynucleotides and thepolynucleotides are associated with the corresponding polypeptides suchthat when a first polypeptide is selected during the screening methodsof the present invention, the polynucleotide sequence that encodes theselected polypeptide is physically linked, or in close proximity, andcan easily be recovered and, for example, sequenced to determine theidentity of the selected polypeptide. In order to achieve this, theplurality of first polynucleotides in a compartment may be such thatthere is on average only one polynucleotide per using the emulsion ofthe present invention.

Further details of avidity based selection methods, which may be used inmethods of the present invention, are described in co-pending GB patentapplication 0114856.8, the contents of which are herein incorporated byreference.

(ii) Catalysis

When selection is for catalysis, the genetic element in eachmicrocapsule may comprise the substrate of the reaction. If the geneticelement encodes a gene product capable of acting as a catalyst, the geneproduct will catalyse the conversion of the substrate into the product.Therefore, at the end of the reaction the genetic element is physicallylinked to the product of the catalysed reaction. When the microcapsulesare combined and the reactants pooled, genetic elements encodingcatalytic molecules can be enriched by selecting for any propertyspecific to the product.

For example, enrichment can be by affinity purification using a molecule(e.g. an antibody) that binds specifically to the product. Equally, thegene product may have the effect of modifying a nucleic acid componentof the genetic element, for example by methylation (or demethylation) ormutation of the nucleic acid, rendering it resistant to or susceptibleto attack by nucleases, such as restriction endonucleases.

Alternatively, selection may be performed indirectly by coupling a firstreaction to subsequent reactions that takes place in the samemicrocapsule. There are two general ways in which this may be performed.First, the product of the first reaction could be reacted with, or boundby, a molecule which does not react with the substrate of the firstreaction. A second, coupled reaction will only proceed in the presenceof the product of the first reaction. An active genetic element can thenbe purified by selection for the properties of the product of the secondreaction.

Alternatively, the product of the reaction being selected may be thesubstrate or cofactor for a second enzyme-catalysed reaction. The enzymeto catalyse the second reaction can either be translated in situ in themicrocapsules or incorporated in the reaction mixture prior tomicroencapsulation. Only when the first reaction proceeds will thecoupled enzyme generate a selectable product.

This concept of coupling can be elaborated to incorporate multipleenzymes, each using as a substrate the product of the previous reaction.This allows for selection of enzymes that will not react with animmobilised substrate. It can also be designed to give increasedsensitivity by signal amplification if a product of one reaction is acatalyst or a cofactor for a second reaction or series of reactionsleading to a selectable product (for example, see Johannsson and Bates,1988; Johannsson, 1991). Furthermore an enzyme cascade system can bebased on the production of an activator for an enzyme or the destructionof an enzyme inhibitor (see Mize et al., 1989). Coupling also has theadvantage that a common selection system can be used for a whole groupof enzymes which generate the same product and allows for the selectionof complicated chemical transformations that cannot be performed in asingle step.

Such a method of coupling thus enables the evolution of novel “metabolicpathways” in vitro in a stepwise fashion, selecting and improving firstone step and then the next. The selection strategy is based on the finalproduct of the pathway, so that all earlier steps can be evolvedindependently or sequentially without setting up a new selection systemfor each step of the reaction.

Expressed in an alternative manner, there is provided a method ofisolating one or more genetic elements encoding a gene product having adesired catalytic activity, comprising the steps of:

-   -   (1) expressing genetic elements to give their respective gene        products;    -   (2) allowing the gene products to catalyse conversion of a        substrate to a product, which may or may not be directly        selectable, in accordance with the desired activity;    -   (3) optionally coupling the first reaction to one or more        subsequent reactions, each reaction being modulated by the        product of the previous reactions, and leading to the creation        of a final, selectable product;    -   (4) linking the selectable product of catalysis to the genetic        elements by either:        -   a) coupling a substrate to the genetic elements in such a            way that the product remains associated with the genetic            elements, or        -   b) reacting or binding the selectable product to the genetic            elements by way of a suitable molecular “tag” attached to            the substrate which remains on the product, or        -   c) coupling the selectable product (but not the substrate)            to the genetic elements by means of a product-specific            reaction or interaction with the product; and    -   (5) selecting the product of catalysis, together with the        genetic element to which it is bound, either by means of a        specific reaction or interaction with the product, or by        affinity purification using a suitable molecular “tag” attached        to the product of catalysis, wherein steps (1) to (4) each        genetic element and respective gene product is contained within        a microcapsule formed from an emulsion of the invention.        (iii) Regulation

A similar system can be used to select for regulatory properties ofenzymes.

In the case of selection for a regulator molecule which acts as anactivator or inhibitor of a biochemical process, the components of thebiochemical process can either be translated in situ in eachmicrocapsule or can be incorporated in the reaction mixture prior tomicroencapsulation. If the genetic element being selected is to encodean activator, selection can be performed for the product of theregulated reaction, as described above in connection with catalysis. Ifan inhibitor is desired, selection can be for a chemical propertyspecific to the substrate of the regulated reaction.

There is therefore provided a method of sorting one or more geneticelements coding for a gene product exhibiting a desired regulatoryactivity, comprising the steps of:

-   -   (1) expressing genetic elements to give their respective gene        products;    -   (2) allowing the gene products to activate or inhibit a        biochemical reaction, or sequence of coupled reactions, in        accordance with the desired activity, in such a way as to allow        the generation or survival of a selectable molecule;    -   (3) linking the selectable molecule to the genetic elements        either by    -   a) having the selectable molecule, or the substrate from which        it derives, attached to the genetic elements, or    -   b) reacting or binding the selectable product to the genetic        elements, by way of a suitable molecular “tag” attached to the        substrate which remains on the product, or    -   c) coupling the product of catalysis (but not the substrate) to        the genetic elements, by means of a product-specific reaction or        interaction with the product;    -   (4) selecting the selectable product, together with the genetic        element to which it is bound, either by means of a specific        reaction or interaction with the selectable product, or by        affinity purification using a suitable molecular “tag” attached        to the product of catalysis.        wherein steps (1) to (4) each genetic element and respective        gene product is contained within a microcapsule formed by an        emulsion of the invention.        (iv) Microcapsule Sorting

The invention provides for the sorting of intact microcapsules wherethis is enabled by the sorting techniques being employed. Microcapsulesmay be sorted as such when the change induced by the desired geneproduct either occurs or manifests itself at the surface of themicrocapsule or is detectable from outside the microcapsule. The changemay be caused by the direct action of the gene product, or indirect, inwhich a series of reactions, one or more of which involve the geneproduct having the desired activity leads to the change. For example,the microcapsule may be so configured that the gene product is displayedat its surface and thus accessible to reagents. Where the microcapsuleis a membranous microcapsule, the gene product may be targeted or maycause the targeting of a molecule to the membrane of the microcapsule.This can be achieved, for example, by employing a membrane localisationsequence, such as those derived from membrane proteins, which willfavour the incorporation of a fused or linked molecule into themicrocapsule membrane. Alternatively, where the microcapsule is formedby phase partitioning such as with water-in-oil emulsions, a moleculehaving parts which are more soluble in the extra-capsular phase willarrange themselves such that they are present at the boundary of themicrocapsule.

In a preferred aspect of the invention, however, microcapsule sorting isapplied to sorting systems which rely on a change in the opticalproperties of the microcapsule, for example absorption or emissioncharacteristics thereof, for example alteration in the opticalproperties of the microcapsule resulting from a reaction leading tochanges in absorbance, luminescence, phosphorescence or fluorescenceassociated with the microcapsule. All such properties are included inthe term “optical”. In such a case, microcapsules can be sorted byluminescence, fluorescence or phosphorescence activated sorting. In ahighly preferred embodiment, fluorescence activated sorting is employedto sort microcapsules in which the production of a gene product having adesired activity is accompanied by the production of a fluorescentmolecule in the capsule. For example, the gene product itself may befluorescent, for example a fluorescent protein such as GFP.Alternatively, the gene product may induce or modify the fluorescence ofanother molecule, such as by binding to it or reacting with it.

(v) Microcapsule Identification

Microcapsules may be identified by virtue of a change induced by thedesired gene product which either occurs or manifests itself at thesurface of the microcapsule or is detectable from the outside asdescribed in section (iv) (Microcapsule Sorting). This change, whenidentified, is used to trigger the modification of the gene within thecompartment. In a preferred aspect of the invention, microcapsuleidentification relies on a change in the optical properties of themicrocapsule resulting from a reaction leading to luminescence,phosphorescence or fluorescence within the microcapsule. Modification ofthe gene within the microcapsules would be triggered by identificationof luminescence, phosphorescence or fluorescence. For example,identification of luminescence, phosphorescence or fluorescence cantrigger bombardment of the compartment with photons (or other particlesor waves) which leads to modification of the genetic element. A similarprocedure has been described previously for the rapid sorting of cells(Keij et al., 1994). Modification of the genetic element may result, forexample, from coupling a molecular “tag”, caged by a photolabileprotecting group to the genetic elements: bombardment with photons of anappropriate wavelength leads to the removal of the cage. Afterwards, allmicrocapsules are combined and the genetic elements pooled together inone environment. Genetic elements encoding gene products exhibiting thedesired activity can be selected by affinity purification using amolecule that specifically binds to, or reacts specifically with, the“tag”.

(vi) Multi-Step Procedure

It will be also be appreciated that according to the present invention,it is not necessary for all the processes of transcription/replicationand/or translation, and selection to proceed in one single step, withall reactions taking place in one microcapsule. The selection proceduremay comprise two or more steps. First, transcription/replication and/ortranslation of each genetic element of a genetic element library maytake place in a first microcapsule. Each gene product is then linked tothe genetic element which encoded it (which resides in the samemicrocapsule). The microcapsules are then broken, and the geneticelements attached to their respective gene products optionally purified.Alternatively, genetic elements can be attached to their respective geneproducts using methods which do not rely on encapsulation. For examplephage display (Smith, G. P., 1985), polysome display (Mattheakkis etal., 1994), RNA-peptide fusion (Roberts and Szostak, 1997) or lacrepressor peptide fusion (Cull, et al., 1992).

In the second step of the procedure, each purified genetic elementattached to its gene product is put into a second microcapsulecontaining components of the reaction to be selected. This reaction isthen initiated. After completion of the reactions, the microcapsules areagain broken and the modified genetic elements are selected. In the caseof complicated multistep reactions in which many individual componentsand reaction steps are involved, one or more intervening steps may beperformed between the initial step of creation and linking of geneproduct to genetic element, and the final step of generating theselectable change in the genetic element.

(vii) Selection by Activation of Reporter Gene Expression In Situ

The system can be configured such that the desired binding, catalytic orregulatory activity encoded by a genetic element leads, directly orindirectly to the activation of expression of a “reporter gene” that ispresent in all microcapsules. Only gene products with the desiredactivity activate expression of the reporter gene. The activityresulting from reporter gene expression allows the selection of thegenetic element (or of the compartment containing it) by any of themethods described herein.

For example, activation of the reporter gene may be the result of abinding activity of the gene product in a manner analogous to the “twohybrid system” (Fields and Song, 1989). Activation might also resultfrom the product of a reaction catalysed by a desirable gene product Forexample, the reaction product could be a transcriptional inducer of thereporter gene. For example arabinose could be used to inducetranscription from the araBAD promoter. The activity of the desirablegene product could also result in the modification of a transcriptionfactor, resulting in expression of the reporter gene. For example, ifthe desired gene product is a kinase or phosphatase the phosphorylationor dephosphorylation of a transcription factor may lead to activation ofreporter gene expression.

(viii) Amplification

According to further aspects of methods of the present invention themethods may comprise the further step of amplifying the geneticelements. Selective amplification may be used as a means to enrich forgenetic elements encoding the desired gene product.

In all the above configurations, genetic material comprised in thegenetic elements may be amplified and the process repeated in iterativesteps. Amplification may be by the polymerase chain reaction (Saiki etal., 1988) or by using one of a variety of other gene amplificationtechniques including; Qβ replicase amplification (Cahill, Foster andMahan, 1991; Chetverin and Spirin, 1995; Katanaev, Kurnasov and Spirin,1995); the ligase chain reaction (LCR) (Landegren et al., 1988; Barany,1991); the self-sustained sequence replication system (Fahy, Kwoh andGingeras, 1991) and strand displacement amplification (Walker et al.,1992).

EXAMPLES Example 1 Conventional Emulsions do not Support EfficientEukaryotic in Vitro Translation

An oil phase formulation comprising 4.5% v/v sorbitan monooleate (Span80, Fluka; 85548) and 0.4% v/v polyoxyethylenesorbitan monooleate (Tween80, Sigma Ultra; P-8074, and 0.05% v/v t-octylphenoxypolyethoxyethanol(Triton-X 100, Sigma), in mineral oil (Sigma; M-3516) (“original CSRmix”) was used to emulsify a RRL expression reaction. All RRL reactionswere performed using the TNT T7 quick coupled transcription/translationsystem (Promega)) expressing firefly luciferase from a plasmid template.A 50 μl expression reaction was set up on ice comprising 80% RRL (v/v),methionine 0.02 mM, and luciferase plasmid template (1 ug). A 40 μlaliquot of this was emulsified by dropwise addition (1 drop per 5 secs)to 100 μl of ice-chilled oil phase under constant stirring (1000 rpm).After addition of the last drop (approximately 20 secs) stirring wascontinued for an additional 3-4 minutes.

Emulsified reactions were incubated at 30° C. for 1 hour and extractedusing ether as described above. Luciferase activity was measured in a96-well plate reader using luciferase assay reagent (Promega). Thenon-emulsified reaction was also extracted with ether to enable justcomparison of activities. This showed expression levels within emulsionto be typically a very low 0.1% that of the non-emulsified reaction.

Using a “reduced” oil phase formulation, comprising 1.5% (v/v) Span 80(SIGMA) in mineral oil (SIGMA), slightly improved yields of up to 0.7%were noted, especially with addition of DTT to 20 μM. Inclusion of DTTusing the original CSR oil mix was extremely detrimental to emulsionformation. Several ratios of aqueous phase to oil phase were tested, andmixing times varied. The conditions described above gave highestactivity with retention of emulsion integrity over the incubationperiod.

Thus emulsification of a RRL reaction using the “classic” emulsionformulations based on Span80 either alone or in combination with Tween80and TritonX-100 resulted in a complete loss of in vitro translationactivity (<1% as expression outside emulsion (Table 2)) as judged byexpression of the enzyme firefly luciferase.

Furthermore, we noted a colour change of the hemin component of the RRLfrom a dark pink to a rusty brown colour within a matter of minutesafter emulsification (see FIG. 1), indicating oxidation of the hemin,presumably attributable to a component of the oil phase.

Example 2 Anti-oxidants do not Restore Efficient Eukaryotic in VitroTranslation

Mammalian translation is regulated, inter alia, by the family of eIF-2αkinases in response to cellular stress (reviewed by Dever (1999), TIBS,24, 398-403). eIF-2α kinases contain a sensor domain and a conservedeIF2 kinase domain. Their function is to phosphorylate eIF-2α (whichshuts down translation) in response to stimuli received through thesensor domain. This serves to protect the cell in circumstances ofcellular stress from wasting resources (protein translation consumeslarge amounts of cellular energy) or as an anti-viral defence bypreventing expression of the viral genome. Four different kinases areknown to respond to different types of stress signals (Table 1).

TABLE 1 Stress-responsive eIF2 kinases Name Activating signal HRIoxidative stress (low heme) PKR viral infection (dsRNA) GCN2 starvation(low amino acids, uncharged tRNA) PERK heat stress, viral infection(unfolded protein)

We reasoned that one of the reasons for the complete shut down intranslation (given the discoloring of the hemin in the RRL) wasoxidative stress caused by the diffusion of oxidative species from theoil into the water-phase.

The component responsible for discoloration and the inhibition oftranslation was identified to be contained in both the Span80 andTween80 surfactant but not in the mineral oil.

We tried to buffer the redox change by providing anti-oxidant orreducing compounds within the oil-phase (BDH, BDA) and/or thewater-phase (DTT, ascorbic acid). Furthermore, we tried to removeoxidative species from the oil-phase by reaction of the oil-phase (themineral oil surfactant mixture) or Span80 alone with(Polystyrylmethyl)trimethylammoniurn borohydride-beads (Novabiochem) orDTT prior to emulsification. Although slowing discoloration (FIG. 1),this yielded only slight improvements in translation efficiencies (Table2). Furthermore, treatment of the oil-phase with borohydride-beadsappeared to reduce the emulsion-forming properties of thesurfactant/oil-phase leading to much destabilized emulsions.

Only DTT (at 20 μM final conc.) produced a slight but consistentimprovement in expression levels within emulsion compared to that ofnon-emulsified reaction (Table 2)). Higher levels of DTT proveddetrimental to the RRL expression system (not shown). In general DTTalso proved destabilizing to the emulsions.

Example 3 Neither cAMP nor 2-AP Enable Efficient Eukaryotic in VitroTranslation

We also attempted a direct inhibition of the eIF2 kinase pathway usingcAMP or 2-aminopurine (2-AP). Although cAMP produced a small improvementwhen used on its own, it proved not-additive with the beneficial effectsof DTT (not shown). Both cAMP and 2-AP are rather unspecific inhibitorsof eIF2 kinases (there are at present no available specific inhibitors)and presumably interfere with other processes within the RRL translationsystem.

Example 4 Neither HSP70 nor HSP90 Enable Efficient Eukaryotic in VitroTranslation

The fact that even high concentrations of DTT did not rescue more thanresidual translation activity suggested to us that other factors thanoxidation must be contributing to translation inhibition. One suspectwas protein denaturation as affected by the emulsification process, i.e.the vigorous mixing of a hydrophilic water phase and hydrophobicoil-phase. Indeed, it has been known for some time that addition of BSA(presumably containing some denatured protein produced byfreeze-thawing) produces a translation shutdown (Matts et al (1993),Biochemistry, 32, 7323-7328), even without emulsification. We confirmedthe results of Matts 93 with and without emulsification. We also triedto buffer the effect by supplementing the RRL lysates with chaperones,in particular HSP70 and HSP90 (SIGMA). Once again this produced onlysmall (non additive) improvements (not shown), which could be equalledor surpassed by the addition of small amounts (5%) of glycerol (notshown), which is known to protect proteins from denaturation.

Example 5 Development of a Novel Oil-Phase and Water-in-Oil Emulsion

Given this low level of activity, efforts were made to identify a noveloil phase formulation with diminished potential to cause oxidativestress or protein denaturation during the emulsification process.

After attempts to improve activity with Span80 based emulsions hadfailed we tested novel surfactants.

Emulsification reactions were performed as described in Example 1, butsubstituting the surfactants used in that reaction (Span 80, Tween 80,and Triton) with silicone-based surfactants. Of these, chemically inertsilicone-based surfactants were found to be successful.

Polysiloxane-polycetyl-polyethylene glycol copolymer (ABIL EM90)(Goldschmidt) used at 4% (v/v) in mineral oil afforded significantimprovements. Emulsifying as described in Example 1, with the exceptionof a reduced (1.5 minute) stirring time, expression levels dramaticallyincreased up to 39% (Table 2) of that of the non-emulsified reaction.This emulsion was stable throughout the incubation period, andmicroscopic visualisation revealed the majority of compartments to havediameters in the range of 2-5 microns (FIG. 2).

TABLE 2 Luciferase activity (in light units (LU)) of luciferaseexpressed in rabbit reticulocyte extract non-emulsified and emulsifiedat two different mixing times (3 min and 4 min) Emul- sified: CSR 1.5%Non mixing emulsion Span80 in emulsified time mix mineral oil 4% Abil−DTT 326789 3 min 945  832 127828 +DTT 231318 N.A*. 1632  78970 %activity  0.2% 0.2%/0.6 39%/34% Back- ground: 249 −DTT 154442 4 min 327 338  40155 +DTT 101702 N.A*.  843  29493 % activity 0.13% 0.15%/0.726%/29% Back- ground: 114 *non-stable emulsion

Example 6 Expression of Human Telomerase in Emulsion using NovelOil-phase

Telomeres are specific DNA structures found at the ends of chromosomesin eukaryotes. They are appended to the 3′ end (left after removal ofthe RNA primer used for DNA replication) by a specialised enzymetelomerase, comprising a RNA component and a protein component. Inhumans the enzyme comprises human telomeric RNA (hTR) and the humantelomerase polypeptide (hTERT) and appends repeated copies of a hexamerrepeat (TTAGGG; SEQ ID NO:1) to the chromosome ends. Telomerase activityhas been implicated in both tumor development and progression as well asin senescence and programmed cell death.

Telomerase has so far not been expressed successfully in functional formin prokaryotes, presumably because it requires specialised chaperones(e.g. HSP 90) and accessory factors both for assembly and function. Butit can be expressed in eukaryotic cells and also in the rabbitreticulocyte lysate in vitro translation system (Weinrich et al (1997),Nat. Genet., 17, 498-502/Beattie et al (1998, 8, 177-180).

The oil phase from Example 5 was tested for expression of functionalhuman telomerase in RRL. Linear T7-driven templates encoding either wthTERT and hTR in tandem cloned downstream of the IRKS of pCITE4a(Novagen) or hTERT cloned into pCITE4a with hTR cloned separately into avariant of pCITE 4a (in which the IRKS was deleted (pCITE-E)) or aninactive deletion mutant of telomerase were prepared by PCRamplification using the respective parental plasmids and primersCITET7ba (5′ GTTTTCCCAGTCACGACGTTG TAA-3′; SEQ ID NO:2) and CITEterfo(5′ 25 CCGGATATAGTTCCTCCTTTCAGC-3′; SEQ ID NO:3).

Expression reactions (50 μl) comprised RRL (80% v/v), telomeraseconstruct (see FIG. 3 legend), methionine (1 mM), telomerase substrate(TS (telomerase substrate) primer (5′ AATCCGTCGAGCAGAGTT-3′; SEQ IDNO:4) (Intergen) 1 μM), PCR enhancer (Promega) (3 μl), 30 dNTPs (25 μM),DTT (20 μM). Emulsions were formed under chilled conditions (on ice)exactly as described above and incubated at 30° C. for 1.5 hours.Emulsions were then extracted with 200 μl chloroform-isoamylalcohol,followed by extraction of aqueous phase with 1 volume phenol-chloroform.

Telomerase reaction products were ethanol precipitated and resuspendedin 20 ul sterile water. A PCR-based TRAP (Kim 94) assay (Intergen) wasthen used to detect telomerase extension products formed in the emulsionTelomerase activity produces a characteristic ladder (with 6 bp spacingfor each repeat) (see FIG. 3).

The results indicated expression of functionally active telomerase fromthe WT template in emulsion. Control experiments either omittingaddition of telomerase gene, or using an inactive deletion mutant gavenegative results (FIG. 3).

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1. A method for increasing the concentration of a nucleic acid moleculecomprising the steps of: (a) forming microcapsules from a water-in-oilemulsion consisting of: a chemically inert silicone-based surfactant, ahydrophobic oil external phase, and a hydrophilic aqueous internal phasecomprising the microcapsules, wherein the microcapsules include anucleic acid molecule, a bead capable of being linked to the nucleicacid molecule, and an aqueous solution comprising components necessaryto perform nucleic acid amplification; (b) amplifying the nucleic acidmolecule in the microcapsules to form amplified copies of the nucleicacid molecule, wherein the amplification employs a thermal cyclingprocess, thereby increasing the concentration of the nucleic acidmolecule in the microcapsules.
 2. The method of claim 1 wherein theamplified copies are captured to the beads in the microcapsules.
 3. Themethod of claim 1 wherein the nucleic acid amplification is performedusing a method selected from the group consisting of Qb replicaseamplification, ligase chain reaction, self sustained sequencereplication, and strand displacement amplification.
 4. The method ofclaim 1 wherein the water-in-oil emulsion comprises a water to oil ratioof 1 to 2.5.
 5. The method of claim 1 wherein the nucleic acid moleculeis genomic DNA or cDNA.
 6. The method of claim 1 wherein the nucleicacid molecule comprises a tag selected from the group consisting ofbiotin, digoxigenin, and 2,4-dinitrophenyl.
 7. The method of claim 1wherein the bead comprises a coating selected from the group consistingof avidin, streptavidin, anti-digoxigenin antibodies, andanti-2,4-dinitrophenyl antibodies.
 8. The method of claim 1 wherein thebead is selected from the group consisting of polystyrene and magneticbeads.
 9. The method of claim 1 wherein the chemically inertsilicone-based surfactant is provided at a v/v concentration range of0.5-20% of surfactant in oil.
 10. The method of claim 1 wherein the beadwith the captured amplification copies is sorted for sequencing.
 11. Themethod of claim 1 wherein a plurality of microcapsules when formed eachcontains on average one or less than one nucleic acid molecule.
 12. Themethod of claim 1 wherein a plurality of microcapsules when formed eachcontains on average between 5 and 1000 nucleic acid molecules.